Use of thymineless death to enrich for doubly auxotrophic mutants of Bacillus megaterium.

Wachsman, J. T. (University of Illinois, Urbana), and L. Hogg. Use of thymineless death to enrich for doubly auxotrophic mutants of Bacillus megaterium. J. Bacteriol. 87:1118-1122. 1964.-When strain KM:T(-), a thymine auxotroph of Bacillus megaterium strain KM, is allowed to undergo thymineless death on a minimal medium, the survivors are greatly enriched in polyauxotrophic mutants. Cells were irradiated with ultraviolet light, grown in the presence of thymidine and a complete amino acid mixture, and then starved for thymidine in the absence of amino acids. Doubly auxotrophic mutants (thymine(-) amino acid(-)) may account for more than 90% of the survivors. The most reproducible results were obtained when sucrose (0.4 m) was added to both growth and starvation media. Although the percentage of mutants among the survivors increases with the time of thymine starvation, the absolute number of double auxotrophs per milliliter decreases. It is probable that the extent of cross-feeding determines both the mutant yield and the mutants types. Substrains of KM:T(-) having additional requirements for each of the following amino acids have been isolated: histidine, threonine, tyrosine, tryptophan, arginine, isoleucine, methionine, serine, and cysteine.